RIG‐I is a key cytosolic sensor that detects RNA viruses through its C‐terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While posttranslational modification has been demonstrated to regulate RIG‐I signaling activity, its significance for the sensing of viral RNAs remains unclear. Here, we first show that the RIG‐I C‐terminal region undergoes deacetylation to regulate its viral RNA‐sensing activity and that the HDAC6‐mediated deacetylation of RIG‐I is critical for viral RNA detection. HDAC6 transiently bound to RIG‐I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG‐I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild‐type mice. These findings underscore the critical role of HDAC6 in the modulation of the RIG‐I‐mediated antiviral sensing pathway. Synopsis Deacetylation of the cytoplasmic RIG‐I viral sensor by HDAC6 is required for viral RNA detection. HDAC6 depletion impairs in vivo responses against RNA but not DNA viruses in mice. HDAC6 interacts with and deacetylates RIG‐I in response to RNA virus infection. Deacetylation of the K909 residue on RIG‐I regulates its viral RNA‐sensing activity. HDAC6−/− mice are highly susceptible to RNA virus infection. Deacetylation of the cytoplasmic RIG‐I viral sensor by HDAC6 is required for viral RNA detection. HDAC6 depletion impairs in vivo responses against RNA but not DNA viruses in mice.